bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2023.07.31.551351

The Crk4-Cyc4 complex regulates G2 phase of apicomplexan endodyogeny

Hawkins, L. M.·Wang, C.·Chaput, D.·Batra, M.·Marsilia, C.·Awshah, D.·Suvorova, E.
View original source ↗Export metadata ↓

Abstract

Division of apicomplexan parasites differs drastically from the division of their host cells. A fraction of apicomplexans divides in the traditional binary mode, such as Toxoplasma gondii in asexual stages, whereas the vast majority instead divide in a multinuclear fashion. Such variety of replication modes and a dearth of conserved conventional regulators have hindered the progress of apicomplexan cell cycle studies. We previously identified five Cdk-related kinases (Crk) involved in endodyogenic division of T. gondii tachyzoites. The current study investigates the roles of a novel essential cell cycle kinase TgCrk4. We identified this kinase cyclin partner and demonstrated that TgCrk4 regulates processes carried out during conventional G2 phase, such as repression of chromosome rereplication and centrosome re-duplication. Accumulation of TgCyc4 in the nucleus and on the centrosomes supported the role of TgCrk4-TgCyc4 complex as a coordinator of chromosome and centrosome cycles in T. gondii. Examination of the TgCrk4-deficient tachyzoites confirmed a cell cycle stop prior to the TgCrk6-regulated spindle assembly checkpoint. Furthermore, we identified an ortholog of the DNA replication licensing factor Cdt1 that was a dominant interactor of the TgCrk4-TgCyc4 complex. T. gondii Cdt1 is highly divergent but preserved critical signature domains and appeared to play a minimal or no role in licensing DNA replication in G1 phase. Functional analyses indicated the primary role of TgCdt1 is in controlling chromosome rereplication and centrosome reduplication. Global phosphoproteome analyses identified immediate TgCrk4 substrates, such as DNA replication licensing factor TgORC4, component of the anaphase-promoting complex TgCdc20, {gamma}-tubulin nucleation factor TgGCP2, and the catalytic subunit of cell cycle phosphatase TgPP2ACA. Importantly, our phylogenetic and structural analyses revealed that the functional TgCrk4-TgCyc4 complex was encoded in the limited group of apicomplexans dividing in a binary fashion. Together with the minimal representation of binary division in Apicomplexa phylum, our findings support the novel view of apicomplexans acquiring binary division to repress ancestral multinuclear mechanisms.

Explore related subjects

cell biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Hawkins, L. M., Wang, C., Chaput, D., Batra, M., Marsilia, C., Awshah, D., Suvorova, E.. 2023-08-02. The Crk4-Cyc4 complex regulates G2 phase of apicomplexan endodyogeny. https://doi.org/10.1101/2023.07.31.551351

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Sequence context and partner availability specify divergent cell fates from a single transcription factor

Transcription factors drive the gene regulatory networks that control developmental transitions, yet which of their many targets a cell uses remains unresolved. Expressing Cebp, Cebp{beta} or Cebp{delta} identically, alone or with Oct4, Sox2, Klf4 and cMyc, gave four outcomes: adipocyte from fibroblasts and neural stem cells, trophectoderm from embryonic stem cells, and myeloid from B cells. Raising Cebp levels changed how many sites it reached, not which sequence it read: clustered motifs maintained somatic enhancers, while spillover onto low-copy sites created de novo enhancers carrying composites of Cebp and cell-type-restricted partners. In fibroblasts, Cebp also redistributed Oct4, Sox2 and Klf4 from early targets, blocking iPSC formation. Supplying the predicted partners rewrote the output: Spi1 redirected Cebp into parallel adipogenic and myeloid programs, via a transient PpargCD45 state using separate enhancers for each; TFAP2C abolished adipogenic conversion. Motif density and partner composites therefore assemble a factor's instructive output in the receiving cell.

cell biology↗
bioRxiv2026

High-content screening identifies FDA-approved activators of mitophagy

Mitochondrial diseases are highly heterogeneous disorders caused by defects in oxidative phosphorylation, for which only limited therapeutic options are available. The elimination of dysfunctional mitochondria via mitophagy activation is an attractive strategy to treat several mitochondrial diseases, irrespective of the underlying genetic defect. To identify pharmacological activators of this pathway, we performed a high-content screening of 1,971 FDA-approved compounds using cellular reporters of TFEB nuclear translocation and mitophagy. This screening identified 294 compounds that promoted TFEB translocation, nine of which increased the number of mitolysosomes without compromising cell viability or damaging mitochondria. Among the selected compounds, Meptazinol and Ponatinib emerged as the top-ranked mitophagy inducers in fibroblasts from patients with complex I and IV deficiency, respectively, and were prioritised for further characterisation. However, they did not rescue the bioenergetic defect, suggesting that the stimulation of mitophagy per se is not sufficient to ameliorate the OXPHOS defect in mitochondrial diseases. Overall, our findings establish a scalable platform for the identification of pharmacological activators of TFEB and identify candidate compounds for therapeutic modulation of mitophagy.

cell biology↗
bioRxiv2026

Matrix Gla Protein deficiency reveals a previously unrecognized mucosal calcification in the mouse airways

Tracheal calcification is a common finding that is considered to have limited clinical significance when detected most often in elderly individuals. Despite its frequent occurrence, the cellular and molecular mechanisms at the origin of tracheal calcification remain largely unexplored. In this context, we aimed to characterize the development of tracheal calcification, and to determine the role of matrix Gla protein (MGP), a major inhibitor of ectopic calcification whose genetic or pharmacologic deficiency is associated with premature tracheal calcification in humans. We found that wild-type mice undergo an unexpected early physiologic calcification of the tracheal cartilage rings beginning around one month after birth. This calcification progresses in a dynamic rostro-caudal sequence through a process involving terminal differentiation of tracheal chondrocytes. These findings challenge the prevailing view that tracheal cartilage is a permanent hyaline cartilage and instead reveals an early and highly organized physiologic calcification process. Furthermore, our findings further identify MGP as a key regulator of calcification within the respiratory tract. Indeed, MGP deficiency not only markedly accelerates tracheal cartilage calcification through a similar pattern and mechanism but also induces a previously unrecognized calcification of the tracheal mucosa, a lesion absent in wild-type mice. These alterations could partly explain the respiratory manifestations observed in MGP-deficient patients having Keutel syndrome, which include dyspnea, cough, stridor, and recurrent infections, symptoms previously being mainly attributed to calcification of the tracheal cartilage rings.

cell biology↗